Hexadecyl Treprostinil crystals and methods for preparation thereof

ABSTRACT

There is provided stable crystalline Form I and Form II of Hexadecyl Treprostinil (C 16 TR) and processes for the preparation thereof. The stable crystalline Form I and Form II of Hexadecyl Treprostinil present advantages in storage, formulation, shipment and handling for commercially considerations.

FIELD OF THE INVENTION

The present invention relates in general to solid forms of prostacyclinderivatives and in particular, to solid crystalline forms of HexadecylTreprostinil and preparation methods thereof.

BACKGROUND OF THE INVENTION

Hexadecyl Treprostinil (C₁₆TR) is a synthetic benzoprostacyclin analogueas the prodrug of Treprostinil. The structures of Treprostinil andHexadecyl Treprostinil are shown in the following Scheme A. HexadecylTreprostinil is of great importance from a pharmacologically point ofview. The inhaled Hexadecyl Treprostinil formulated in lipidnanoparticles is a long acting pulmonary vasodilator providingsignificantly lower plasma concentrations than infused Treprostinil(Tyvaso®) for treating pulmonary arterial hypertension (PAH), and can beapplied for less toxicity, better tolerability and more convenientdosing schedules, e.g., Pulm. Pharmacol. Ther., 49, 104-111, DrugResearch, 68, 605-614, and US 2015/148414.

The synthesis processes of Hexadecyl Treprostinil had been disclosed byDrug Research, 68, 605-614 and US 2017/0320813. However, these reportsrevealed that the prepared Hexadecyl Treprostinil is an off-white waxysolid. It is well known that a waxy solid is an amorphous form of activepharmaceutical ingredients (API). The thermal stability, purity andhomogeneity of an amorphous API are not satisfactory comparing to acrystalline one. Besides, a waxy solid is usually provided with highviscosity, and is difficult for commercially handling to avoid stickinglost on container. Since there is no other report disclosing thecrystalline form of Hexadecyl Treprostinil, the benefits of theconventional crystallization methods for preparing a stable HexadecylTreprostinil crystal cannot be evaluated based on the prior artreference.

Consequently, there is an urgent demand for the preparation of a stablecrystalline form of Hexadecyl Treprostinil. A stable HexadecylTreprostinil crystal having fixed physicochemical properties can provideconstant operating parameters, e.g., solubility for regularpharmaceutical formulation and steady bioabsorbability forpharmacological treatment. Additionally, a stable Hexadecyl Treprostinilcrystal can also present advantages in storage, formulation, handlingand shipment and for commercially considerations.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides a stable solidform of Hexadecyl Treprostinil crystal Form I, and processes for thepreparation thereof.

In one embodiment, the present invention provides a method for preparinga crystalline Form I of Hexadecyl Treprostinil, which comprisesdissolving Hexadecyl Treprostinil in a first solvent selected from thegroup consisting of ethyl ether, isopropyl ether, methyl tert-butylether, methyl isobutyl ketone, ethyl acetate, isopropyl acetate,toluene, xylene, acetone, dichloromethane, 1,4-dioxane, tetrahydrofuran,methanol, ethanol, propanol, butanol, benzyl alcohol, dimethylsulfoxide, dimethylacetamide, dimethylformamide, and mixtures thereof toform a homogenous solution; lowering the temperature and/or adding asecond solvent selected from the group consisting of acetonitrile,water, and mixtures thereof to the homogenous solution; and stirringuntil a precipitate is formed.

In one embodiment, the present invention provides a crystalline Form Iof Hexadecyl Treprostinil having an X-ray powder diffraction (XRPD)pattern exhibiting its six strongest characteristic peaks at thefollowing 2θ reflection angles: 3.3±0.2°, 6.6±0.2°, 14.2±0.2°,18.9±0.2°, 21.3±0.2°, and 22.5±0.2°.

In one embodiment, the present invention provides a crystalline Form Iof Hexadecyl Treprostinil having a differential scanning calorimetry(DSC) thermogram pattern comprising an endothermic peak with a peakonset temperature of approximately 52.2±1° C. and a peak maximum ofapproximately 54.5±1° C.

According to one aspect, the present invention provides a stable solidform of Hexadecyl Treprostinil crystal Form II, and processes for thepreparation thereof.

In one embodiment, the present invention provides a method for preparinga crystalline Form II of Hexadecyl Treprostinil, which comprisesdissolving Hexadecyl Treprostinil in a third solvent selected from thegroup consisting of ethyl ether, isopropyl ether, methyl tert-butylether, methyl isobutyl ketone, ethyl acetate, isopropyl acetate,toluene, xylene, acetone, dichloromethane, 1,4-dioxane, tetrahydrofuran,methanol, ethanol, propanol, butanol, benzyl alcohol, dimethylsulfoxide, dimethylacetamide, dimethylformamide, and mixtures thereof toform a homogenous solution; lowering the temperature and/or adding afourth solvent selected from the group consisting of pentane, hexane,heptane, octane, nonane, decane, cyclopentane, cyclohexane,cycloheptane, and mixtures thereof to the homogenous solution; andstirring until a precipitate is formed.

In one embodiment, the present invention provides a crystalline Form 11of Hexadecyl Treprostinil having an XRPD pattern exhibiting its sixstrongest characteristic peaks at the following 2θ reflection angles:3.41±0.2°, 6.1±0.2°, 9.4-0.2°, 20.3±0.2°, 21.6±0.2°, and 23.4±0.2°.

In one embodiment, the present invention provides a crystalline Form IIof Hexadecyl Treprostinil having a DSC thermogram pattern comprising anendothermic peak with a peak onset temperature of approximately 54.6±1°C. and a peak maximum of approximately 56.9±1° C.

The present invention provides solid forms of Hexadecyl Treprostinilcrystal Form I and Form II, which can stable storage at room temperaturewithout crystalline form transformation for commercially handling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an X-ray powder diffraction (XRPD) pattern of HexadecylTreprostinil crystal Form I.

FIG. 2 shows a differential scanning calorimetry (DSC) thermogrampattern of Hexadecyl Treprostinil crystal Form I.

FIG. 3 shows a Fourier Transform Infrared (FTIR) spectrum of HexadecylTreprostinil crystal Form I.

FIG. 4 shows an X-ray powder diffraction (XRPD) pattern of HexadecylTreprostinil crystal Form II.

FIG. 5 shows a differential scanning calorimetry (DSC) thermogrampattern of Hexadecyl Treprostinil crystal Form II.

FIG. 6 shows a Fourier Transform Infrared (FTIR) spectrum of HexadecylTreprostinil crystal Form II.

DETAILED DESCRIPTION OF THE INVENTION

Hexadecyl Treprostinil Crystal Form I and Preparation Thereof

In an embodiment of the present invention, the method for preparingHexadecyl Treprostinil crystal Form I comprises the steps of:

-   (a) dissolving crude Hexadecyl Treprostinil in a first solvent    selected from the group consisting of ethyl ether, isopropyl ether,    methyl tert-butyl ether, methyl isobutyl ketone, ethyl acetate,    isopropyl acetate, toluene, xylene, acetone, dichloromethane,    1,4-dioxane, tetrahydrofuran, methanol, ethanol, propanol, butanol,    benzyl alcohol, dimethyl sulfoxide, dimethylacetamide,    dimethylformamide, and mixtures thereof to form a homogenous    solution;-   (b) lowering the temperature and/or adding a second solvent selected    from the group consisting of acetonitrile, water, and mixtures    thereof to the homogeneous solution;-   (c) stirring until a precipitate is formed;-   (d) filtering out the precipitate thereby isolating the Hexadecyl    Treprostinil crystal Form I; and-   (e) optionally drying the Hexadecyl Treprostinil crystal Form I.

In the present invention, the first solvent used to dissolve the crudeHexadecyl Treprostinil is selected from the group consisting of ethylether, isopropyl ether, methyl tert-butyl ether, methyl isobutyl ketone,ethyl acetate, isopropyl acetate, toluene, xylene, acetone,dichloromethane, 1,4-dioxane, tetrahydrofuran, methanol, ethanol,propanol, butanol, benzyl alcohol, dimethyl sulfoxide,dimethylacetamide, dimethylformamide, and mixtures thereof, preferablyethanol and propanol. The volume of the first solvent depends on thetypes of the solvents used and may be about 0.5 ml to about 100 ml,preferably about 1 ml to about 50 ml, and more preferably about 2 ml toabout 20 ml or about 5 ml to about 10 ml, per 1 g of the crude HexadecylTreprostinil. The crude Hexadecyl Treprostinil can be dissolved in thefirst solvent at a temperature ranging from about 0° C. to about 80° C.,preferably from about 10° C. to so about 60° C., and more preferablyfrom room temperature to about 40° C.

In one embodiment of the present invention, the temperature of thehomogenous solution is lowered to a temperature ranging from about −30°C. to about 50° C., preferably from about −20° C. to about 40° C., andmore preferably from about −10° C. to about 30° C.

The selection of the second solvent is the key to determine whether aHexadecyl Treprostinil crystal Form I can be formed. In a preferredembodiment, the volume of the second solvent selected from the groupconsisting of acetonitrile, water, and mixtures thereof depends on thetypes of the solvents used and may be about 0.5 ml to about 200 ml,about 1 ml to about 150 ml, or about 2 ml to about 100 ml, per 1 ml ofthe first solvent. The second solvent can be added at a temperatureranging from about −30° C. to about 50° C., preferably from about −20°C. to about 40° C., and more preferably from about −10° C. to about 30°C.

In one embodiment of the present invention, the precipitation of thecrystal may be performed at a temperature ranging from about −30° C. toabout 50° C., preferably from about −20° C. to about 40° C., and morepreferably from about −10° C. to about 30° C.

In one embodiment of the present invention, the step of filtering outthe precipitate comprises using the second solvent or a mixture of thefirst solvent and the second solvent to wash the precipitate. In themixing solvent, the ratio of the first solvent and the second solventmay be about 1:1 to about 1:100, preferably about 1:10 to about 1:50.

In one embodiment of the present invention, the Hexadecyl Treprostinilcrystal Form I has an X-ray powder diffraction (XRPD) pattern exhibitingits five strongest characteristic peaks at the following 2θ reflectionangles: 3.3±0.2°, 6.6±0.2°, 14.2±0.2°, 18.9±0.2°, 21.3±0.2°, and22.5±0.2°. In a preferred embodiment, the XRPD pattern of HexadecylTreprostinil crystal Form I further comprises characteristic peaks atthe following 2θ reflection angles: 13.8±0.2°, 15.3±0.2°, 16.9±0.2°,17.8±0.2°, 19.8±0.2°, 20.6±0.2°, 20.9±0.20, 24.4±0.2°, and 24.8±0.2°.More preferably, the XRPD pattern of Hexadecyl Treprostinil crystal FormI is consistent with FIG. 1. The particular data of HexadecylTreprostinil crystal Form I is shown in Table 1.

TABLE 1 2θ angle (°) d value (Å) relative intensity (%) 3.3 26.6 100.06.6 13.4 40.1 9.8 9.0 4.5 13.1 6.8 2.8 13.8 6.4 7.9 14.2 6.2 46.0 15.35.8 11.3 16.3 5.4 6.7 16.9 5.2 19.7 17.8 5.0 16.0 18.9 4.7 92.8 19.8 4.514.5 20.6 4.3 8.2 20.9 4.2 20.2 21.3 4.2 32.2 22.5 4.0 27.8 24.4 3.627.7 24.8 3.6 19.4 26.2 3.4 4.5 27.3 3.3 2.3 27.8 3.2 2.1 28.8 3.1 1.529.9 3.0 1.6 31.2 2.9 1.2 32.5 2.8 1.5 34.0 2.6 2.5 36.2 2.5 1.8 36.82.4 3.4 39.7 2.3 2.7

In one embodiment, the present invention provides a HexadecylTreprostinil crystal Form I having an XRPD pattern substantially asshown in FIG. 1.

In one embodiment, the present invention provides a HexadecylTreprostinil crystal Form I having a differential scanning calorimetry(DSC) thermogram pattern comprising an endothermic peak with a peakonset temperature of approximately 52.2±1° C. and a peak maximum ofapproximately 54.5±1° C. In a preferred embodiment, the presentinvention provides a Hexadecyl Treprostinil crystal Form I having a DSCthermogram pattern substantially as shown in FIG. 2.

In one embodiment, the present invention provides a HexadecylTreprostinil crystal Form I having a 1% KBr Fourier Transform Infrared(FTIR) spectrum comprising peaks, in terms of cm⁻¹, at 3445±4 cm⁻¹,3402±4 cm⁻¹, 2958±4 cm⁻¹, 2919±4 cm⁻¹, 2871.4 cm⁻¹, 2854±4 cm⁻¹, 1733±4cm⁻¹, 1606±4 cm⁻¹, 1585=4 cm⁻¹, 1474±4 cm⁻¹, 1443±4 cm⁻¹, 1416.4 cm⁻¹,1374±4 cm⁻¹, 1357±4 cm⁻¹, 1331±4 cm⁻¹, 1309±cm⁻¹, 1271±4 cm⁻¹, 1247±4cm⁻¹, 1229±4 cm⁻¹, 120214 cm⁻¹, 1167±4 cm⁻¹, 1148±4 cm⁻¹, 1122±4 cm⁻¹,1088±4 cm⁻¹, 1039±4 cm⁻¹, 1023±4 cm⁻¹, 1000±4 cm⁻¹, 982±4 cm⁻¹, 968±4cm⁻¹, 917±4 cm⁻¹, 889±4 cm⁻¹, 784±4 cm⁻¹, 772±4 cm⁻¹, 7344 cm⁻¹, 719±4cm⁻¹, 708±4 cm⁻¹. In a preferred embodiment, the present inventionprovides a Hexadecyl Treprostinil crystal Form I having a 1% KBr FTIRspectrum substantially as shown in FIG. 3.

Due to the organic solvent system used in the method of the presentinvention, the precipitated Hexadecyl Treprostinil crystal Form Ipossesses compact solid characteristics and thus is easy to be filteredout. The residual solvents can be easily removed under high vacuum atroom temperature. Moreover, the dried Hexadecyl Treprostinil crystalForm I with granular characteristics is much easier to weight forcommercially handling comparing with the waxy solid form of HexadecylTreprostinil with high viscosity.

In addition, Hexadecyl Treprostinil crystal Form I is a stablecrystalline form, which shows good stability, with no other crystallineforms or degraded products of impurities at room temperature for sixmonths. Moreover, the assay of Hexadecyl Treprostinil crystal Form I canbe kept between about 98.0% to about 102.0% even after thirty-six monthsof placement under normal storage temperature (about 5° C. to about −20°C.).

Hexadecyl Treprostinil Crystal Form II and Preparation Thereof

In an embodiment of the present invention, the method for preparingHexadecyl Treprostinil crystal Form II comprises the steps of:

-   (a) dissolving crude Hexadecyl Treprostinil in a third solvent    selected from the group consisting of ethyl ether, isopropyl ether,    methyl tert-butyl ether, methyl isobutyl so ketone, ethyl acetate,    isopropyl acetate, toluene, xylene, acetone, dichloromethane,    1,4-dioxane, tetrahydrofuran, methanol, ethanol, propanol, butanol,    benzyl alcohol, dimethyl sulfoxide, dimethylacetamide,    dimethylformamide, and mixtures thereof to form a homogenous    solution;-   (b) lowering the temperature and/or adding a fourth solvent selected    from the group consisting of pentane, hexane, heptane, octane,    nonane, decane, cyclopentane, cyclohexane, cycloheptane, and    mixtures thereof to the homogeneous solution;-   (c) stirring until a precipitate is formed;-   (d) filtering out the precipitate thereby isolating the Hexadecyl    Treprostinil crystal Form II; and-   (e) optionally drying the Hexadecyl Treprostinil crystal Form II.

In the present invention, the third solvent used to dissolve the crudeHexadecyl Treprostinil is selected from the group consisting of ethylether, isopropyl ether, methyl ten-butyl ether, methyl isobutyl ketone,ethyl acetate, isopropyl acetate, toluene, xylene, acetone,dichloromethane, 1,4-dioxane, tetrahydrofuran, methanol, ethanol,propanol, butanol, benzyl alcohol, dimethyl sulfoxide,dimethylacetamide, dimethylformamide, and mixtures thereof, preferablyethyl acetate and toluene. The volume of the third solvent depends onthe types of the solvents used and may be about 0.5 ml to about 100 ml,preferably about 1 ml to about 50 ml, and more preferably about 2 ml toabout 20 ml or about 1 ml to about 10 ml, per 1 g of the crude HexadecylTreprostinil. The crude Hexadecyl Treprostinil can be dissolved in thethird solvent at a temperature ranging from about 0° C. to about 80° C.,preferably from about 10° C. to about 60° C., and more preferably fromroom temperature to about 40° C.

In one embodiment of the present invention, the temperature of thehomogenous solution is lowered to a temperature ranging from about −30°C. to about 50° C., preferably from about −20° C. to about 40° C., andmore preferably from about −10° C. to about 30° C.

The selection of the fourth solvent is the key to determine whether aHexadecyl Treprostinil crystal Form II can be formed. In a preferredembodiment, the volume of the fourth solvent selected from the groupconsisting of pentane, hexane, heptane, octane, nonane, decane,cyclopentane, cyclohexane, cycloheptane, and mixtures thereof depends onthe types of the solvents used and may be about 0.5 ml to about 200 ml,about 1 ml to about 150 ml, or about 2 ml to about 100 ml, per 1 ml ofso the third solvent. The fourth solvent can be added at a temperatureranging from about −30° C. to about 50° C., preferably from about −20°C. to about 40° C., and more preferably from about −10° C. to about 30°C.

In one embodiment of the present invention, the precipitation of thecrystal may be performed at a temperature ranging from about −30° C. toabout 50° C., preferably from about −20° C. to about 40° C., and morepreferably from about −10° C. to about 30° C.

In one embodiment of the present invention, the step of filtering outthe precipitate comprises using the fourth solvent or a mixture of thethird solvent and the fourth solvent to wash the precipitate. In themixing solvent, the ratio of the third solvent and the fourth solventmay be about 1:1 to about 1:100, preferably about 1:10 to about 1:50.

In one embodiment of the present invention, the Hexadecyl Treprostinilcrystal Form II has an XRPD pattern exhibiting its six strongestcharacteristic peaks at the following 2θ reflection angles: 3.4±0.2°,6.1±0.2°, 9.4±0.2°, 20.3±0.2°, 21.6±0.2°, and 23.4±0.2°. In a preferredembodiment, the XRPD pattern of Hexadecyl Treprostinil crystal Form IIfurther comprises characteristic peaks at the following 2θ reflectionangles: 7.0±0.2°, 9.0±0.2°, 12.2±0.2°, 12.7±0.2°, 17.5±0.2°, 18.0±0.2°,18.5±0.2°, 19.1±0.2°, and 19.4±0.2°. More preferably, the XRPD patternof Hexadecyl Treprostinil crystal Form II is consistent with FIG. 4. Theparticular data of Hexadecyl Treprostinil crystal Form II is shown inTable 2.

TABLE 2 2θ angle (°) d value (Å) relative intensity (%) 3.4 26.0 100.04.5 19.6 2.2 6.1 14.5 45.1 7.0 12.6 22.0 7.7 11.5 3.9 9.0 9.8 27.8 9.49.4 85.5 10.0 8.8 3.6 10.6 8.3 13.9 17.2 7.7 22.6 12.7 7.0 29.9 13.9 6.43.8 14.2 6.2 3.9 14.9 5.9 5.2 15.5 5.7 17.2 16.1 5.5 18.1 16.7 5.3 6.217.5 5.1 25.4 18.0 4.9 30.8 18.5 4.8 79.3 19.1 4.6 33.8 19.4 4.6 30.320.3 4.4 39.9 20.9 4.2 19.8 71.6 4.1 40.6 22.6 3.9 9.4 23.4 3.8 81.925.4 3.5 9.3 27.7 3.2 3.6 28.8 3.1 3.1 30.9 2.9 2.3 37.3 2.4 2.1 40.02.3 2.8 43.3 2.1 2.1

In one embodiment, the present invention provides a HexadecylTreprostinil crystal Form II having an XRPD pattern substantially asshown in FIG. 4.

In one embodiment, the present invention provides a HexadecylTreprostinil crystal Form II having a DSC thermogram pattern comprisingan endothermic peak with a peak onset temperature of approximately54.6±1° C. and a peak maximum of approximately 56.9±1° C. In a preferredembodiment, the present invention provides a Hexadecyl Treprostinilcrystal Form II having a DSC thermogram pattern substantially as shownin FIG. 5.

In one embodiment, the present invention provides a HexadecylTreprostinil crystal Form II having a 1% KBr FTIR spectrum comprisingpeaks, in terms of cm⁻¹, at 3515±4 cm⁻¹, 3443±4 cm⁻¹, 3291±4 cm⁻¹,3034±4 cm⁻¹, 29534 cm⁻¹, 2922±4 cm⁻¹, 2851±4 cm⁻¹, 1769±4 cm⁻¹, 1730±4cm⁻¹, 1605±4 cm⁻¹, 1584±4 cm⁻¹, 1469±4 cm⁻¹, 1456±4 cm⁻¹, 1437±4 cm⁻¹,1395±4 cm⁻¹, 1371±4 cm⁻¹, 347±4 cm⁻¹, 1330±4 cm⁻¹, 1311±4 cm⁻¹, 1295±4cm⁻¹, 1286±4 cm⁻¹, 1271±4 cm⁻¹, 1259±4 cm⁻¹, 1235±4 cm⁻¹, 1218±4 cm⁻¹,1209±4 cm⁻¹, 1189±4 cm⁻¹, 1171±4 cm⁻¹, 1148±4 cm⁻¹, 1115±4 cm⁻¹, 1070±4cm⁻¹, 1053±4 cm⁻¹, 1030±4 cm⁻¹, 1019±4 cm⁻¹, 989±4 cm⁻¹, 946±4 cm⁻¹,926±4 cm⁻¹, 9094 cm⁻¹, 893±4 cm⁻¹, 878±4 cm⁻¹, 790±4 cm⁻¹, 765±4 cm⁻¹,726±4 cm⁻¹, 704±4 cm⁻¹. In a preferred embodiment, the present inventionprovides a Hexadecyl Treprostinil crystal Form II having a 1% KBr FTIRspectrum substantially as shown in FIG. 6.

Due to the organic solvent system used in the method of the presentinvention, the precipitated Hexadecyl Treprostinil crystal Form IIpossesses compact solid characteristics and thus is easy to be filteredout. The residual solvents can be easily removed under high vacuum atroom temperature. Moreover, the dried Hexadecyl Treprostinil crystalForm II with granular characteristics is much easier to weight forcommercially handling comparing with the waxy solid form of HexadecylTreprostinil with high viscosity.

In addition, Hexadecyl Treprostinil crystal Form II is a stablecrystalline form, which shows good stability, with no other crystallineforms or degraded products of impurities at room temperature for sixmonths. Moreover, the assay of Hexadecyl Treprostinil crystal Form IIcan be kept between about 98.0 to about 102.0% even after thirty-sixmonths of placement under normal storage temperature (about 5° C. toabout −2° C.).

EXAMPLES

X-ray Powder Diffraction (XRPD) Analysis: The XRPD patterns werecollected on a Bruker D2 PHASER diffractometer with fixed divergenceslits and 1D LYNXEYE detector. The samples (ca. 100 mg) were flatlyplaced on a sample holder. The prepared samples were analyzed over a 20range from 20 to 50° with step size of 0.02 degrees and step time of 1second using CuK_(α) radiation at a power of 10 mA and 30 kV. TheCuK_(β) radiation was removed by a divergent beam nickel filter.

Differential Scanning Calorimetry (DSC) Analysis: The DSC thermogrampatterns were collected on a TA DISCOVERY DSC25 instrument. The samples(ca. 5 mg) were weighed into an aluminum pan with a crimping closedaluminum lid. The prepared samples were analyzed from 10° C. to 100° C.at scan rate of 10° C./min under a flow of nitrogen (ca. 50 ml/min). Themelting temperature and heat of fusion were calibrated by indium (In)before measurement.

Fourier Transform Infrared (FTIR) Analysis: The FTIR spectra werecollected on a Perkin Elmer SPECTRUM 100 instrument. The samples weremixed with potassium bromide (KBr) in an approximately 1:100 ratio (w/w)using an agate mortar and pestle. The mixture was compressed in a pelletdie at a pressure of about 10 to 13 tonnes for 2 minutes. The resultingdisk was scanned 4 times versus a collected background from 4000 cm⁻¹ to650 cm⁻¹ at a resolution of 4 cm⁻¹. The data was baseline corrected andnormalized.

Example 1

Preparation of Crude Hexadecyl Treprostinil

2-(((1R,2R,3aS,9aS)-2-hydroxy-1-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalen-5-yl)oxy)aceticacid (40.0 g, 102.4 mmol) was dissolved in 600 ml dimethylformamide andfollowed by addition of 42.0 g potassium carbonate with 72.0 gI-iodohexadecane, and stirred at 60° C. for 1 hour. Afterwards, thereaction mixture was slowly cooled to 10° C., and 650 ml water and 650ml ethyl so acetate with 50.0 g magnesium sulfate were added to thereaction mixture for extraction. The extraction solution was evaporatedoff under vacuum at room temperature to get crude product. The crudeproduct was then purified by chromatography on silica gel using amixture of hexane and ethyl acetate as a gradient eluent to obtain 58.6g off-white waxy solid (crude Hexadecyl Treprostinil).

Example 2

Preparation of Hexadecyl Treprostinil Crystal Form I

Crude Hexadecyl Treprostinil (1.00 g, from Example 1) and propanol (5mil) were heated to 40° C. for dissolution and then cooled to roomtemperature. Water (5 ml) was added slowly dropwise and the mixture wasstirred in ice water bath for 18 hours until solid precipitationoccurred. Afterwards, the resulting suspension was filtered and rinsed,and then dried under high vacuum at room temperature for 24 hours togive 0.92 g Hexadecyl Treprostinil crystal Form I. The XRPD, DSC andFTIR results are the same as shown in FIG. 1, FIG. 2 and FIG. 3.

Example 3

Preparation of Hexadecyl Treprostinil Crystal Form I

Crude Hexadecyl Treprostinil (1.01 g, from Example 1) and ethanol (6mil) were heated to 40° C. for dissolution and then cooled to roomtemperature. Acetonitrile (30 ml) was added slowly dropwise and themixture was stirred in ice water bath for 18 hours until solidprecipitation occurred. Afterwards, the resulting suspension wasfiltered and rinsed, and then dried under high vacuum at roomtemperature for 24 hours to give 0.90 g Hexadecyl Treprostinil crystalForm I. The XRPD, DSC and FTIR results are the same as shown in FIG. 1,FIG. 2 and FIG. 3.

Example 4

Preparation of Hexadecyl Treprostinil Crystal Form I

Crude Hexadecyl Treprostinil (1.0) g, from Example 1) and toluene (5 ml)were heated to 40° C. for dissolution. Acetonitrile (50 ml) was addedslowly dropwise and the mixture was stirred in ice water bath for 20hours until solid precipitation occurred. Afterwards, the resultingsuspension was filtered and rinsed, and then dried under high vacuum atroom temperature for 24 hours to give 0.91 g Hexadecyl Treprostinilcrystal Form I. The XRPD, DSC and FTIR results are the same as shown inFIG. 1, FIG. 2 and FIG. 3.

Example 5

Preparation of Hexadecyl Treprostinil Crystal Form II

Crude Hexadecyl Treprostinil (1.01 g, from Example 1) and ethyl acetate(1 ml) were heated to 40° C. for dissolution and then cooled to roomtemperature. N-hexane (30 ml) was added slowly dropwise and the mixturewas stirred in ice water bath for 18 hours until solid precipitationoccurred. Afterwards, the resulting suspension was filtered and rinsed,and then dried under high vacuum at room temperature for 24 hours togive 0.88 g Hexadecyl Treprostinil crystal Form II. The XRPD, DSC andFTIR results are the same as shown in FIG. 4, FIG. 5 and FIG. 6.

Example 6

Preparation of Hexadecyl Treprostinil Crystal Form II

Crude Hexadecyl Treprostinil (1.00 g, from Example 1) and toluene (3 ml)were heated to 40° C. for dissolution and then cooled to roomtemperature. N-heptane (40 ml) was added slowly dropwise and the mixturewas stirred in ice water bath for 24 hours until solid precipitationoccurred. Afterwards, the resulting suspension was filtered and rinsed,and then dried under high vacuum at room temperature for 24 hours togive 0.85 g Hexadecyl Treprostinil crystal Form II. The XRPD, DSC andFTIR results are the same as shown in FIG. 4, FIG. 5 and FIG. 6.

Example 7

Preparation of Hexadecyl Treprostinil Crystal Form II

Crude Hexadecyl Treprostinil (1.01 g, from Example 1) and methyltert-butyl ether (3 ml) were heated to 40° C. for dissolution. N-pentane(30 ml) was added slowly dropwise and the mixture was stirred in icewater bath for 18 hours until solid precipitation occurred. Afterwards,the resulting suspension was filtered and rinsed, and then dried underhigh vacuum at room temperature for 24 hours to give 0.85 g HexadecylTreprostinil crystal Form II. The XRPD, DSC and FTR results are the sameas shown in FIG. 4, FIG. 5 and FIG. 6.

What is claimed is:
 1. A crystalline Form I of Hexadecyl Treprostinil,characterized by having an X-ray powder diffraction (XRPD) patterncomprising its six strongest characteristic peaks at the following 2θreflection angles: 3.3±0.2°, 6.6±0.2°, 14.2±0.2°, 18.9±0.2°, 21.3±0.2°,and 22.5±0.2°.
 2. The crystalline Form I of Hexadecyl Treprostinil ofclaim 1, wherein the XRPD pattern further comprises characteristic peaksat the following 2θ reflection angles: 13.8±0.2°, 15.3±0.2°, 16.9±0.2°,17.8±0.2°, 19.8±0.2°, 20.6±0.2°, 20.9±0.2°, 24.4±0.2°, and 24.8±0.2°. 3.The crystalline Form I of Hexadecyl Treprostinil of claim 1, wherein theXRPD pattern is substantially shown in FIG.
 1. 4. The crystalline Form Iof Hexadecyl Treprostinil of claim 1, further having a differentialscanning calorimetry (DSC) thermogram pattern comprising an endothermicpeak with a peak onset temperature of approximately 52.2±1° C. and apeak maximum of approximately 54.5±1° C.
 5. The crystalline Form I ofHexadecyl Treprostinil of claim 4, wherein the DSC thermogram pattern issubstantially shown in FIG.
 2. 6. The crystalline Form I of HexadecylTreprostinil of claim 1, further having a 1% KBr Fourier transforminfrared (FTIR) spectrum comprising peaks, in terms of cm⁻¹, at 3445±4cm⁻¹, 3402±4 cm⁻¹, 2958±4 cm⁻¹, 2919±4 cm⁻¹, 2871±4 cm⁻¹, 2854±4 cm⁻¹,1733±4 cm⁻¹, 1606±4 cm⁻¹, 1585±4 cm⁻¹, 1474±4 cm⁻¹, 1443±4 cm⁻¹, 1416±4cm⁻¹, 1374±4 cm⁻¹, 1357±4 cm⁻¹, 1331±4 cm⁻¹, 1309±4 cm⁻¹, 1271±4 cm⁻¹,1247±4 cm⁻¹, 1229±4 cm⁻¹, 1202±4 cm⁻¹, 1167±4 cm⁻¹, 1148±4 cm⁻¹, 1122±4cm⁻¹, 1088±4 cm⁻¹, 1039±4 cm⁻¹, 1023±4 cm⁻¹, 1000±4 cm⁻¹, 982±4 cm⁻¹,968±4 cm⁻¹, 917±4 cm⁻¹, 889±4 cm⁻¹, 784±4 cm⁻¹, 7724 cm⁻¹, 734±4 cm⁻¹,719±4 cm⁻¹, 708±4 cm⁻¹.
 7. The crystalline Form I of HexadecylTreprostinil of claim 6, wherein the FTIR spectrum is substantiallyshown in FIG.
 3. 8. A method for preparing the crystalline Form I ofHexadecyl Treprostinil according to claim 1, which comprises the stepsof: dissolving Hexadecyl Treprostinil in a first solvent selected fromthe group consisting of ethyl ether, isopropyl ether, methyl tert-butylether, methyl isobutyl ketone, ethyl acetate, isopropyl acetate,toluene, xylene, acetone, dichloromethane, 1,4-dioxane, tetrahydrofuran,methanol, ethanol, propanol, butanol, benzyl alcohol, dimethylsulfoxide, dimethylacetamide, dimethylformamide, and mixtures thereof toform a homogenous solution, lowering the temperature and/or adding asecond solvent selected from the group consisting of acetonitrile,water, and mixtures thereof to the homogenous solution; and stirringuntil a precipitate is formed.
 9. The method of claim 8, furthercomprising the steps of: adding the second solvent or a mixture of thefirst solvent and the second solvent for rinsing the precipitate;filtering out the precipitate thereby isolating the crystalline Form Iof Hexadecyl Treprostinil; and optionally drying the crystalline Form Iof Hexadecyl Treprostinil.
 10. A crystalline Form II of HexadecylTreprostinil, characterized by having an XRPD pattern comprising its sixstrongest characteristic peaks at the following 2θ reflection angles:3.4±0.2°, 6.1±0.2°, 9.4±0.2°, 20.3±0.2°, 21.6±0.2°, and 23.4±0.2°. 11.The crystalline Form II of Hexadecyl Treprostinil of claim 10, whereinthe XRPD pattern further comprises characteristic peaks at the following20 reflection angles: 7.0±0.2°, 9.0±0.2°, 12.2±0.2°, 12.7±0.2°,17.5±0.2°, 18.0±0.2°, 18.5±0.2°, 19.1±0.2°, and 19.4±0.2°.
 12. Thecrystalline Form II of Hexadecyl Treprostinil of claim 10, wherein theXRPD pattern is substantially shown in FIG.
 4. 13. The crystalline FormII of Hexadecyl Treprostinil of claim 10, further having a DSCthermogram pattern comprising an endothermic peak with a peak onsettemperature of approximately 54.6±1° C. and a peak maximum ofapproximately 56.9±1° C.
 14. The crystalline Form II of HexadecylTreprostinil of claim 13, wherein the DSC thermogram pattern issubstantially shown in FIG.
 5. 15. The crystalline Form II of HexadecylTreprostinil of claim 10, further having a 1% KBr FTIR spectrumcomprising peaks, in terms of cm⁻¹, at 3515±4 cm⁻¹, 3443±4 cm⁻¹, 3291±4cm⁻¹, 3034±4 cm⁻¹, 2953±4 cm⁻¹, 2922±4 cm⁻¹, 2851±4 cm⁻¹, 1769±4 cm⁻¹,1730±4 cm⁻¹, 1605±4 cm⁻¹, 1584±4 cm⁻¹, 1469±4 cm⁻¹, 1456±4 cm⁻¹, 1437±4cm⁻¹, 1395±4 cm⁻¹, 1371±4 cm⁻¹, 1347±4 cm⁻¹, 1330±4 cm⁻¹, 1311±4 cm⁻¹,1295±4 cm⁻¹, 1286±4 cm⁻¹, 1271±4 cm⁻¹, 1259±4 cm⁻¹, 1235±4 cm⁻¹, 1218±4cm⁻¹, 1209±4 cm⁻¹, 1189±4 cm⁻¹, 1171±4 cm⁻¹, 1148±4 cm⁻¹, 1115±4 cm⁻¹,1070±4 cm⁻¹, 1053±4 cm⁻¹, 1030±4 cm⁻¹, 1019±4 cm⁻¹, 989±4 cm⁻¹, 946±4cm⁻¹, 926±4 cm⁻¹, 909±4 cm⁻¹, 893±4 cm⁻¹, 878±4 cm⁻¹, 790±4 cm⁻¹, 765±4cm⁻¹, 726±4 cm⁻¹, 704±4 cm⁻¹.
 16. The crystalline Form II of HexadecylTreprostinil of claim 15, wherein the FTIR spectrum is substantiallyshown in FIG.
 6. 17. A method for preparing the crystalline Form II ofHexadecyl Treprostinil according to claim 10, which comprises the stepsof: dissolving Hexadecyl Treprostinil in a third solvent selected fromthe group consisting of ethyl ether, isopropyl ether, methyl tert-butylether, methyl isobutyl ketone, ethyl acetate, isopropyl acetate,toluene, xylene, acetone, dichloromethane, 1,4-dioxane, tetrahydrofuran,methanol, ethanol, propanol, butanol, benzyl alcohol, dimethylsulfoxide, dimethylacetamide, dimethylformamide, and mixtures thereof toform a homogenous solution; lowering the temperature and/or adding afourth solvent selected from the group consisting of pentane, hexane,heptane, octane, nonane, decane, cyclopentane, cyclohexane,cycloheptane, and mixtures thereof to the homogenous solution; andstirring until a precipitate is formed.
 18. The method of claim 17,further comprising the steps of: adding the fourth solvent or a mixtureof the third solvent and the fourth solvent for rinsing the precipitate;filtering out the precipitate thereby isolating the crystalline Form IIof Hexadecyl Treprostinil; and optionally drying the crystalline Form IIof Hexadecyl Treprostinil.